Desensitization is a mechanism that plays an important role in turning off receptor-mediated signal transduction pathways. The goal of the proposed studies is to define the molecular basis for two processes associated with the rapid phase of desensitization of G-protein coupled receptors (GPRs), namely receptor/G-protein (R/G) uncoupling and receptor internalization. The studies will use muscarinic cholinergic receptors (mAChRs), particularly the m2 mAChRs, as a model, as enhanced or diminished receptor signalling through mAChR subtypes has been suggested to play critical roles in several diseases, including Alzheimer's and Parkinson's diseases, and the cardiovascular pathologies associated with Chagas' disease. The hypothesis that underlies this proposal is that agonists induce phosphorylation of the mAChR on defined serine/threonine (S/T) and this initiates several distinct downstream events that cause R/G uncoupling and internalization. In other systems, arrestins bind to phosphorylated GPRs to cause R/G uncoupling and guide GPRs to clathrin coated pits for endocytosis. Significant progress has been made in understanding phosphorylation-dependent events associated with desensitization of the mR mAChR, however, it is unclear how phosphorylation leads to R/G uncoupling and internalization. Compelling data suggest that arrestins do not play a major role in internalization of the m2 mAChR, leading us to question the putative role of arrestins in R/G uncoupling. Aim one will test the hypothesis that arrestins cause uncoupling of m2 mAChR from G-proteins. Over expression of arrestin binding proteins and anti-sense constructs will be used to deplete endogenous arrestins and ascertain their effects on R/G uncoupling. Aim two will define the mechanisms and role of phosphorylation-dependent, arrestin-independent internalization of mAChRs. Potential roles of caveolae and mAChR palmitoylation will be examined A search will be made for interacting proteins that may guide the receptors to the endocytic machinery. Aim 3 will test the hypothesis that S/T-rich clusters analogous to those identified in the m2 mAChR might be newly recognized motifs that have a common regulatory role in regulation of GPRs. The S/T residues in two clusters in the third cytoplasmic domains of the Gq-coupled m3 and the Gi-coupled m4 mAChRs will be mutated and the effects on agonist-dependent phosphorylation and desensitization will be assessed. The results will provide new insights into novel events involved in turning off the signals from mAChRs.